Prostaglandin 1,15-lactones of the F series from the nudibranch

Prostaglandin 1,15-lactones of the F series from the nudibranch mollusk Tethys fimbria. Guido Cimino, Antonio Crispino, Vincenzo Di Marzo, Aldo Spinel...
1 downloads 0 Views 740KB Size
J. Org. Chem. 1991,56, 2907-2911 for C14H79BOlaa). Anal. Calcd for C14H79B010:C, 66.85; H, 9.92. Found: C, 66.19; H, 10.15. Reaction of 18 with Ethanolamine. Formation of 20. A solution of 18 (38 mg,44 pmol) in CH2Ch (2 mL) was treated with excess ethanolamine (ca. 0.1 mL), and the resulting solution was maintained at 23 OC for 24 h. The solution was then diluted with ether and washed with water and brine. The organic material was dried (MgSOJ and concentrated. The crude product was purified by chromatography (silica gel, 240-400 mesh, EtOAchexane (5:1)), giving 32 mg (80%)of 20 as a clear oil: 'H NMR (CDCl,, 490 MHz) 6 5.73 (m, 1 H), 5.40 (br s, 1 H), 5.14-4.92 (m, 6 H), 4.56 (br d, J = 13.5, Hz, 1 H), 4.03 (dd, J = 9.1, 4.9 Hz, 1 H), 3.83-3.70 (m, 4 H), 3.65-3.50 (m, 2 H), 3.45-3.20 (m, 3 H), 3.42 (8,3 H), 3.39 (8, 3 H), 3.30 (8, 3 H), 3.02 (ddd, J = 4.2,8.8, 11.3 Hz, 1 H), 2.71-2.63 (m, 2 H), 2.55 (m, 1 H), 2.40-1.95 (m, 8 H), 1.95-1.15 (m, 17 H), 1.68 (8, 3 H), 1.55 (8,3 H), 1.15-0.80 (m, 20 H); 13C NMR (63 MHz, CDC13) 171.1, 167.6, 165.5, 137.3, 136.7, 129.1, 125.5, 115.6, 95.8, 84.3, 76.7, 74.9, 74.0, 73.7, 72.6, 70.8, 69.5, 61.8, 57.6, 56.8, 56.7, 56.2, 55.8, 50.1, 42.9, 41.0, 39.6, 36.2,35., 35.1, 33.7,33.0,31.4,31.2,30.8,28.1,26.3, 26.2, 26.2,25.0, 22.1,20.2,16.4,15.7,15.3,15.3,14.6,13.5,9.8; IR (film)3403,2936, 1734,1638,1444,1196 cm-'; MS (FAB) mle 915.6202 (915.6120 calcd for CmH81N2BO12),897, 687, 309. (22S)-Dihydro FK-506 (17).19 A solution of 18 (9.5 mg, 11 pmol) in THF (2.0 mL) was treated with H 2 0 (3.0 mL), and the resulting solution was maintained at 23 OC for 2 days. This solution was then diluted with saturated aqueous sodium bicarbonate and extracted with EtOAc. The organic material was dried (K2C03)and concentrated. The crude isolate was purified by chromatography (silica gel, 240-400 mesh, THF-hexanes (40:60)),giving 3.7 mg of 17 (42%) and 2.7 mg of recovered 18: 'H NMR (490 MHz, CDC13) 6 5.78 (m, 1 H), 5.30 (br s, 0.5 H), 5.22 (br s, 0.5 H), 5.20-4.92 (m, 5 H), 4.65 (br s, 1 H), 4.43 (br d, J = 7.6 Hz, 1 H), 3.95-3.80 (m, 13 H), 3.78-3.65 (m, 1 H), 3.65-3.52 (m, 2 H), 3.50-3.20 (m, 5 H), 3.41, 3.38,3.37, 3.32, 3.30 (s,3 X OCH3 for both major and minor rotamera), 3.01 (m, 1 H), 2.84 (m, 0.7 H), 2.70-1.95 (m, 9 H), 1.95-1.20 (m, 14 H), 1.66, 1.64 (8, CH=CHCHS for both major and minor rotamers), 1.54, 1.48 (8,CH-CH CH3 for both major and minor rotamers) 1 . 2 0 . 8 0 (m, 3 3 H); '3c NMR (63 MHz, CDCl,; data given for both major and minor rotamera) 6 195.97,195.82,169.32,169.21,165.65,165.06, 137.48, 136.42,135.86,132.72,131.66,128.95,126.69,126.22,115.72, 90.49, 97.11, 84.31, 78.17, 76.81,75.52, 73.96, 73.84, 73.69, 73.40, 56.26, 56.11, 72.90,71.81, 71.02, 70.06, 70.55,57.02,56.64,56.58,

2907

52.61,49.40,48.97,44.58,44.11,44.00,40.85,39.61,39.50,37.05,

36.85, 35.91, 35.11, 35.05, 34.82,34.08, 33.97,33.02,32.71,32.61, 34.41,30.82, 29.70,27.35,26.85,26.71, 26.05,24.71, 24.63,21.47, 20.67, 16.47, 16.29, 15.77,15.38, 14.62, 14.30,10.50,9.44; IR ( f h ) 3455,2928,1733,1642,1453,1089cm-'; MS (FAB) mle 828.4909 (828.4876 calcd for C&H71012NNa),578. Reductive Amination of (22S)-Dihydro FK-506. A solution of 18 (20 mg, 25 pmol) and dry methanol (0.5 mL) was treated with benzylamine (3.20 pL, 29 pmol), and NaBHSCN (ca 50 mg) at 23 "C for 24 h. At this time, the solution waa diluted with water (10 mL) and extracted with EtOAc. The extracts were dried (MgS04)and concentrated. The crude material was purified by chromatography (silica gel, 240-400 mesh, EtOAc-hexanes (51)), giving 6.6 mg (40%) of 13 as a clear oil. Reduction of FK-506 with NaBH(OAc),. A solution of FK-506 (0.117 g, 0.146 mmol) and THF (1.75 mL) was treated with NaBH(OAc), (0.154 g, 0.730 mmol) and HOAc (0.35 mL) at 23 "C for 2 h. At this time, the solution was diluted with H20 and extracted with EtOAc. The combined extracts were washed with saturated aqueous sodium bicarbonate and brine. The organic material was dried (MgS04)and concentrated. The crude material was purified by chromatography(silica gel, 240-400mesh, 6040 hexanes-THF), giving 9.0 mg of 2019 (7.7%)and 81 mg of 17 (69%). Prepared in this fashion, 17 contains ca. 10% (as evidenced by the appearance of unassignable signals at 6 5.95 (br s) and 5.50 (br s)) of an unknown impurity. Nevertheless, this material was used successfully in the transformations outlined in this paper.

Acknowledgment. This research was supported by PHS Grant AI 16943. An N S F Postdoctoral Fellowship (CHE-8907478) t o M.J.F. as well as a n NIH Postdoctoral Fellowship to A.V. are gratefully acknowledged. We also thank Dr. R. S. Volante of t h e Merck Laboratories for providing spectra of authentic 21 a n d for several helpful discussions. N M R spectra were obtained through t h e auspices of the Northeast regional NSF/NMR Facility at Yale University, which was supported by NSF Chemistry Division Grant CHE 7916210. Supplementary Material Available: NMR spectra for compounds 4,9, 17, and 19-21 (6 pages). Ordering information is given on any current masthead page.

Notes Prostaglandin 1,lbLactones of the F Series from the Nudibranch Mollusc Tethys fimbria Guido Cimino, Antonio Crispino, Vincenzo Di Marzo, Aldo Spinella,; and Guido Sodanot

Istituto per la Chimica di Molecole di Interesse Biologic0 del CNR, Via Toiano, 6 Arco Felice (NA),Italy Received June 26, 1990 We have recently reported for the f i t time t h e natural occurrence of prostaglandin l,l5-lactones of the E series (1-3) in the nudibranch Tethys fimbria.' More recently,2 we have found that these lactones are biosynthesized from free prostaglandins in t h e mantle of t h e mollusc a n d are converted back into t h e prostaglandins upon detachment 'Current address: Dipartimento di Fiaica, UniversitA di Salemo,

84081 Baronissi (SA), Italy.

0022-3263/91/1956-2907$02.50/0

of t h e cerata (body appendices) during the behavioral defense mechanism known as autotomy. We describe now the isolation and structure characterization of prostaglandin 1,15-ladones of the F series from the same mollusc a n d from its egg masses. I n addition, 4, which was not detected previously,' has also been isolated by HPLC and its structure established by comparison with standard PGE2-1,15-1actone 11-acetate. PGF2,-1,15-lactone 11-acetate (5) and PGF,-1,15lactone 11-acetate (6) were isolated from the mantles and cerata of t h e mollusc in t h e relative amounts reported in Table I. Comparison of their 'H N M R spectra (Experimental Section) with those of 1-3l and with the published spectrum of synthetic PGFh-1,15-lactoneS (7) suggested (1) Cimino, G.; Spinella, A.; Sodano,G. Tetrahedron Lett. 1989,30, 3589. (2) Cimino, G.; Crispino, A.; Di Marzo,V.; Sodano,G.; Spinella, A.; Villani, G. Experientia 1991, 47, 56.

0 1991 American Chemical Society

97

Notes

2908 J. Org. Chem., Vol. 56, No.8, 1991 0

~~~

~~~

RO"'

~~

20

1 R=H 4 R-AC

OR1

2 R=H 3 R-AC

QR1

0

5 8

R l = H ; &=Ac 7 & = & = H R1=&=Ac o R1=H;Rz=Ac;9-epi

6 R l = H ; Rz=Ac io R1=H;&=Ac;sepi

the gross structures 5 and 6 for the two compounds. In order to firmly establish the regie and stereochemistry at C-9 and C-11, synthetic 4' was reduced with NaBH4 to afford two isomeric products in a relative ratio of ca. 51, the minor one being undistinguishable from 5 by TLC and 'H NMR spectroscopy, thus assigning the position of the acetyl group and the stereochemistry a t C-11. Furthermore, acetylation of 5 afforded a diacetyl derivative (8) that proved to be identical by TLC and 'H NMR comparison to the diacetyl derivative prepared from synthetic 7," thus confirming the structure and stereochemistry of 5. The major NaBH, reduction product of 4 is by inference 9epi-PGFk-l,15-lactone 11-acetate (9). Similarly, N&H4 reduction of 3 afforded 6 as the minor product and 9epi-PGF,-l,l5-lactone 11-acetate (10) in the same ratio as the reaction products of 4. Egg masses of T. fimbria were collected either in the field or in a laboratory tank where T. fimbria specimens were preserved alive for several days. Extraction and usual purification procedures allowed the isolation from the egg masses of a complex mixture of PGF2a-and PGF,-1,15lactones fatty acid esters (PLFE), while the lactones isolated from the mantles and cerata were absent (Table I). A reversed-phase HF'LC trace of the ester mixture is shown in Figure 1. Part of the ester mixture was submitted to methanolysis (Na2C03,MeOH), which resulted in the production of two compounds (ca. 1:l ratio) coeluting in HPLC with standards of PGFk- and PGF,-methyl esters, PGF2,-1,15lactone (71, and a mixture of fatty acid methyl esters. The structure of PGF%-methyl ester was confirmed by TLC and 'H NMR comparison with an authentic sample, while the structure of PGF,-methyl ester was derived by comparison of its 'H NMR and mass spectral data with those of PGFk-methyl ester. Most of the fatty acid methyl esters were identified by gas chromatography (Experimental Section), the major components being palmitic, cis-5,8,11,14,17-eicosapentaenoic, and ciS-4,7,10,13,16,19docosahexaenoic acid methyl esters. Preliminary information on the constitution of the PLFE was gained from 'H NMR spectra of the crude mixture. The presence of two carbinol protons (6 4.19 and 3.97) in a ca. 1:4 ratio having the characteristic chemical (3) Andersen, N. H.; Bor-Sheng Lin, Biochemistry 1985, 24, 2338. (4) Bundy, G. 4.; Petereon, D. C.; Comette, J. C.; Miller, W. L.; Spilman, C. H., Wilks, J. W. J. Med. Chem. 1983,26,1089.

Table I. Distribution and Amounto of the V d o u r Prostaglandin l,l5-Lactonee in the Anatomical Sections of T.fimbria anatomical &ion comd mantleb ceratab ovotestisc egg massd 1 99 221 2 89 245 3 97 124 4 2 10 6 60 30 6 199 139 PLFE' 950 1600

OMicrograms per gram of dry weight tissue. See the Experimental Section for the quantitation method of compounds 1-6. The dry weights of the various sections of a medium-sized specimen were the following: mantle, 2 g; cerata, 2 g; ovotestis, 0.15 g. The dry weight of an egg mass is ca. 0.9 g. bMean of three specimens. Most of 3 and 4 are transformed into PGAs- and PGAZl,lBlactones, respectively, during the isolation and purification procedures.' CMeanof four specimens. dMean of 15 egg masses. eCrude mixture of PGF,- and PGFh-l,15-lactone fatty acid esters. Table 11. Main Structural Features of the PGFL- and PGF~-l,l5-lactoneFatty Acid Ester Fractions Recovered by TLC (1-3) and HPLC (A-H) main fatty acids fraction prostaglandin 1,lblactone 11-acyl-PGFJ satd/monounsatd 1 9-acyl-PGFL satd/ monounsatd 2 9-acvl-PGFL 9-a&l-PGFG po1yunsatd 9-acyl-PGFh Cm6 9-acyl-PGFS, C14a 9-acyl-PGFh polyunsatd Il-acyl-PGF,/ C22S Cmr 11-acyl-PGFk 9-acyl-PGFk CP26 9-acyl-PGFh C16a ll-acyl-PGF,/ Cia+ and/or Ciw; ll-acyl-PGFh/9-acylC16a and/or PGFS,/9-acyl-PGFk H 9-acyl-PGFk Cl6a C

lb

i

kJ

nmmumln' ' -0

Figure 1. HPLC absorbance (205 nm) profile of the PLFE mixture from 5". fimbria egg masses (Novopak '2-18 4-pm analytical column; CH,0H/H20, 946; 0.8 mL/min). Peak height is not representative of the actual relative abundance since polyunsaturated fatty acyl residues have a higher tm than saturated ones.

shift and shape of H-9 and H-11 of PGFk, together with the presence of a C-11-acylated proton (6 4.90; 1:l with the H-9), suggested that the mixture consisted of PGF-1,15lactones acylated at C-9 (predominant) or C-11. A gross fractionation of the mixture has been achieved by preparative TLC, which yielded three main fractions (1-3, Table

Notes

J. Org. Chem., Vol. 56, No. 8,1991 2909

II), while several peaks of the reversed-phase HPLC shown in Figure 1were isolated (A-H, Table II). Although several

latter, a role in mollusc reproduction seems reasonable.

HPLC peaks were still mixturee, the main structural features of the major components of the fractions were inferred from their 'H NMR and mass spectra with the aid of the criteria discussed below, which were derived by comparative analysis of the spectra and by comparison with the spectra of 5, 6, and 8. In the 'H NMR spectra of the C-11 acylated derivatives (minor components of the mixture), H-9 resonates at 6 4.19 while H-11 resonates at 6 4.90; H-13 and H-14 resonate as two close double doublets at 6 5.88 (J 5.9 and 15.8 Hz) and 5.86 ( J 8.2 and 15.8 Hz). These values are very close to those of 5 and 6. In the C-9 acylated derivatives (major components of the mixture) the H-11 resonates at 6 3.97 while H-9 resonates at 6 5.20, overlapped with H-15; H-13 and H-14 resonate as well as separated double doublets at 6 5.78 (J8.9 and 15.8 Hz) and 5.96 (J6.5 and 15.8 Hz). The presence of polyunsaturated acyl residues was evidenced by large resonances at 6 5.38 (olefinic protons) and 2.84 (methylenes between two double bonds) in a ca. 1:l ratio. In addition, the terminal methyl group of eicosapentaenoic and docosahexaenoic acyl residues resonates at ca. 6 0.97 and was distinguishable from that of the PGFk residue, which, when present, also absorbed at a similar chemical shift value in the 9-acyl derivatives, while in the 11-acyl derivatives this resonance was shifted to higher fields (6 0.95). In the E1 mass spectra of several HPLC fractions, molecular ions were detected that, in conjunction with the presence of the large fragment arising by the loss of the fatty acid(s) from the molecular ion ( m / z 318 and 316 for the PGF2a- and PGF3,-1,15-lactone derivatives, respectively) and with those originating by loss of water from these latter and from the molecular ion, aided in the identification of the fatty acyl residues linked to the PGF-lactones. The combined evidence allows assignment of the structural features reported in Table I1 to the main components of the HPLC fractions. It is noteworthy that in several fractions the prostaglandin lactones have been found esterified by their fatty acid precursors, namely arachidonic or cis-5,8,11,14,17-eicosapentaenoicacid. The PLFEs are absent in the mantle and cerata, where the other lactones have been found. In order to ascertain where these compounds are stored and then transferred to the eggs, anatomical sections of T. fimbria were made and analyzed by TLC. The PLFE mixture was found only in the ovotestis (hermaphrodite gland), a gland where the egg masses are produced (Table I). Interestingly, PLFEs were not found in immature T. fimbria specimens. This fact would suggest a specific role for PLFE's in the egg production, in their development, or in their protection, in relation either to fish predation or to pathogens. A defensive role against fish predation seems unlikely since the PLFE mixture, as well as the PGF derivatives 5 and 6, were not toxic in the Gambusia affinis bioassay, while 1-3 were found active.2 A role, for example, as chemical release factor for ovulation appears more likely, since it has been reported that prostaglandins appear to stimulate egg production in several molluscs.6 In this respect, the functions of PGE- and PGF-1,15-lactones in T. fimbria appear to be distinct, also because of their different anatomical location: for the former, a double role in chemical defense and precursor of PG free acids in the contraction of the cerata have been proposed;2 for the

Experimental Section

(5) For a review see: Stanley-Samueleon, D. W. Biol. Bull. 1987,173, 92.

General Procedures. HPLC analyses were performed on a chromatographequipped with two pumps and a W-via detector. NMR spectra were recorded at 500 MHz. GLC analyses were performed with capillary column FS SE-30-CB-0.5 (25m length, 0.32-m i.d.). Isolation of Prostaglandin Lactones from Mantles and Cerata. In an early extraction procedure' 26 T.fimbria specimens were deprived of the internal organs, and the resulting mantles and cerata were extracted with acetone (1L X 3) over 18 h at room temperature. The acetone was removed under vacuum at 40 OC, and the remaining water was extracted with diethyl ether (100 mL x 3). After treatment with anhydrous Na2S04,the solvent was evaporated to afford an extract (2.29 g) that was chromatographed on a Si gel column and eluted with CHC13and increasing amounts of CH30H and then on p-Porasil HPLC (n-hexane/ EtOAc, 91) to afford 1-3 as previously described.' Acetate 4 (0.8 mg) was isolated as a faster moving peak during the HPLC purification of the fractions containing 3 and identified by comparison of its 'H NMR spectrum with that of an authentic specimen.' Fractions of the CHC13/CH30H column containing 5 and 6 exhibited a characteristic red spot when sprayed with Ce(S01)2/H2SOIon Si gel TLC (R, 0.35 and 0.30, respectively; C6H6/diethyl ether, 7:3). These fractions were pooled (431 mg), chromatographed on a Si gel column, and eluted with c& and increasing amounts of diethyl ether to afford an enriched fraction (17 mg). Preparative HPLC of this fraction on p-Porasil (nhexane/EtOAc, 82) yielded 2 mg of 5 and 2 mg of 6. Prostaglandin F1,1,154actone 11-acetate (5): MS, m/z (%) 378 (M+;0.6), 318 (M+ - AcOH, base peak), 300 (44), 231 (27), 191 (SO),173 (42); 'H NMR 6 (CDC13;assignments made by 'H-'H COSY) 6 5.88 (dd, J 6.2, 16.0 Hz; 1H, H-14), 5.81 (dd, J 8.4, 16.0; 1 H, H-13), 5.53 (dt, J 4.5, 10.7; 1 H, H-6), 5.49 (m; 1H, H-5), 5.18 (dt, J 6.1, 8.1; 1 H, H - E ) , 4.90 (m; 1 H, H-ll), 4.20 (m; 1 H, H-9), 2.03 (8;3 H, acetyl methyl), 0.88 (bt, J6.2; 3 H, H-20). Prostaglandin Fs.1,lElactone 11-acetate (6): MS, m/z (%) 376 (M+, 3), 342 (5), 316 (M+ - AcOH, 46), 307 (29), 298 (base peak),247 (68),229 (701,163 (69);'H-NMR 6 (CDCl,; assignments made by 'H-lH COSY) 5.90 (dd, J 6.0, 15.8 Hz; 1 H, H-14), 5.84 (dd, J8.0, 15.8; 1 H, H-13), 5.54 (dt, J4.9, 10.1; 1 H, H-6), 5.49 (m; 1H, H-18), 5.42 (m; 1H, H-5), 5.30 (m; 1H, H-17), 5.20 (dt, J 6.1, 7.7; 1 H, H-15), 4.90 (m; 1 H, H-ll), 4.20 (m; 1 H, H-9), 2.04 (8; 3 H, acetyl methyl), 0.96 (t, J 7.6; 3 H, H-20). An improved extraction procedure was used in more recent preparations. Typically, mantles and cerata were separately extracted by mixing the tissue in a mortar with sand (Merck; ca. 5 g/animal) and acetone (10 mL/animal). After the tissue was triturated, more acetone was added and the mixture was sonicated for 5 min. The acetone was decanted, and the sonication was repeated for five times with fresh acetone. The pooled acetone extracts were then concentrated under vacuum, and the aqueous residue was extracted with diethyl ether, dried on Na2S04,and chromatographed on a Si gel column (C&s and increasing amounts of diethyl ether). Fractions were monitored by TLC and submitted to preparative HPLC on a p-Porasil column eluted with n-hexanelEt0Ac (85:15; flow rate 1 mL/min). The recovery of PG-lactones was higher than that of the previous extraction method and close to the values estimated by the quantitation method reported below. Quantitation of t h e Prostaglandin Lactones i n t h e Tissues. Each tissue was extracted with 5% AcOH in H20 (l:l, v/v) containing 15 MMindomethacin, and the extracts were purified on Sep-pak cartridges (Waters), washed with 10% MeOH/85% H20/5% AcOH (5 mL), and eluted with MeOH (5 mL). The eluates were monitored by HPLC on a Spherisorb 5-pm column (ODs-2, 4.5 X 25 mm) and eluted with a 90-min gradient from 30 to 70% CH3CN/0.1% CF3COOH in H20/0.1% CF3COOH. UV absorbance was monitored at 205 nm, and quantitation was made by using prostaglandin lactones previously purified from T.fimbria (detection limit 1 pg). Acquisition, integration, and calibration were performed with use of a Maxima 820 chromatography work station (Waters Associates).

2910 J. Org. Chem., Vol. 56, No.8, 1991 NaB& Reduction of 3 and 4. To 3.6 mg of synthetic4*' PG&-l,lB-lactone ll-acetate (4) dissolved in 0.5 mL of MeOH was added 2 mg of NaBH,. After 5 min at room temperature, 5 mL of CHSCOOHwas added and the solvents were evaporated under NP. The reaction mixture was loaded on an analytical Si gel TLC plate (C6H6/diethylether 82) and the mixture of 5 and 9 eluted together with diethyl ether (2.5 mg). The mixture was separated by HPLC on p-Porasil (n-hexane/EtOAc, 8515; flow rate 2 mL/min) to afford, in order of increasing polarity, 9 (1.5 mg) and 5 (ca. 0.3 mg). 9-Epiprostaglandin Fh 1,lClactone 11-acetate(9): MS, m/z318 (M+- AcOH; 151,300 (base peak), 289 (51,274 (9), 243 (8),213 (8), 191 (19); 'H NMR 6 (CDC13)5.89 (dd, J 6.2,16.0 Hz; 1H, H-14), 5.79 (dd, J 8.5,16.0; 1H, H-13), 5.64 (dt, J 5.2,10.8; 1 H, H-6), 5.40 (dt, J6.1, 10.0; 1 H, H-5), 5.14 (dt, J 6.3,8.1; 1 H, H-15),4.98 (dt, J6.0,8.3; 1H, H-ll),4.05 (m; 1H, H-9), 2.01 (e; 3 H, acetyl methyl), 0.88 (t, 6.6; 3 H, H-20). Reduction of 2.0 mg of natural PGE3-l,15-lactonell-acetate (3) under similar conditions with purification as above yielded 10 and 6 in a 5:l ratio, as judged by HPLC. 9-Epiprortaglandin FS, 1,lS-lactone 1l-acetate (10): MS, m / z 376 (M+; 2.5), 316 (M+ - AcOH; base peak), 307 (271,298 (23),247 (W),229 (85),211 (52), 171 (48); 'H NMR 6 (CDClJ 5.91 (dd, J 6.2, 15.8 Hz; 1 H, H-14), 5.83 (dd, J 8.2,15.8; 1 H, H-13), 5.65 (dt, J5.2,lO.h 1H, H-6), 5.48 (m; H-18), 5.41 (m; 1H, H-5), 5.27 (m; 1H, H-17), 5.16 (at,J 6.2, 7.9; 1 H, H-151, 4.97 (dt, J 5.9,8.2; 1H, H-11), 4.05 (m; 1H, H-9), 2.01 (8; 3 H, acetyl methyl), 0.95 (t, J 7.5; 3 H, H-20). Acetylation of 5 and 7. Synthetic PGFk,-1,15-lactone(7; 2 mg) or natural PGFb-l,15-lactone ll-acetate (5; 0.5 mg) was dissolved in 0.5 mL of anhydrous pyridine, and 0.2 mL of acetic anhydride was added. The mixture was kept at room temperature for 18h, 1mL of MeOH waa added, and the solvents were removed under a N2 stream to afford the same diacetate (8). Prostaglandin Fk 1,15-laatone9,ll-diacetate (8): MS, m/z (%) 420 (M+,0.5), 360 (M+- AcOH l),318 (3), 300 (M+- 2 AcOH, base peak), 257 (3), 243 (5), 229 (61,227 (5), 213 (lo), 174 (91,173 (8); 'H NMR 6 (CDCl3) 5.90 (dd, J 6.6, 16.0 Hz; 1H, H-14), 5.78 (dd, J 8.7, 16.0; 1 H, H-13), 5.46 (dt, J4.4,10.8; 1H, H-6), 5.39 (dt, J 6.0, 10.0; 1H, H-5), 5.19 (m; 1H, H-9),5.17 (dt, J 6.4,8.1; 1H, H-15),4.89 (m; 1 H, H-11),2.08 and 2.02 (two s;3 H each, acetyl methyls), 0.88 (t, J 7.0; 3 H, H-20). Isolation of Prostaglandin 1,lS-hctone Fatty Acid Esters (PLFE) from Egg Massee and Ovotestis. Thirty freshly laid egg masses from several specimens of T. fimbria were frozen at -80 "C and subsequently extracted with sand and acetone as described above. Aliquots of the ethereal extract (1.5 g) were submitted,when needed, to preparative Si gel TLC (C&/diethyl ether, 82), and the large band at R, 0.6 & 0.1 was eluted with diethyl ether (48 mg). Part of the purified PLFE mixture (7 mg) was submitted to preparative Si gel TLC (n-hexane/EtOAc, 7:3) to give three bands (1-3; Table 11) weighing 1.3,1.3, and 0.5 mg, respectively, which were characterized by 'H N M R spectroscopy (CDC13). Diagnostic resonances were as follows: fraction 1,6 5.97 (dd, J 6.5,15.8 Hz), 5.88 (dd, J5.9, 15.8), 5.86 (dd, J8.2,15.8), 5.78 (dd, J8.9, 15.8), 4.90 (m), 4.19 (m), 3.97 (m) (4.m3.97 = 1:l); fraction 2, 6 5.97 (dd, J 6.5,15.8 Hz), 5.81 (dd, J 8.9,15.8), 3.97 (m), 0.97 (t,J 7.41, 0.88 (t, J 6.9); fraction 3, 6 5.97 (dd, J 6.6.15.9 Hz), 5.81 (dd, J 8.9, 15.9), 5.38 (m), 3.97 (m), 2.83 (m), 0.975 (t, J 7.31, 0.972 (t, J 7.3) (0.9750.972 1:l). Part of the purified PLFE mixture (25 mg) was fractionated on a reversed-phase HPLC using repeated l-mg runs on a Novopak 4pm analytical column (Waters; CH30H/H20,946; flow rate 0.8 mL/min) and monitoring the eluate at 205 nm. The eight major components (A-H, Figure 1)were collected to afford A, 1 mg; B, 2 mg; C, 3.7 mg; D, 0.5 mg; E, 1.1mg; F, 1.2 mg; G, 0.8 mg;and H, 3.2 mg and analyzed by 'H NMR spectra (CDCIJ and 600, EIMS (assignments,Table 11). Fraction A m/z 618 (M+), 316, 298; 6 5.97 (dd, J 6.5, 15.5 Hz), 5.81 (dd, J 8.5, 15.5), 5.38 (m), 3.97 (m), 2.82 (m), 0.974 (t, J 7.5), 0.970 (t, J 7.5). Fraction B m / z 544 (M+),526,316,298; 6 5.97 (dd, J 6.5, 15.8 Hz),5.81 (dd, J8.9, 15.8), 3.96 (m),0.97 (t, J7.5), 0.88 (t,J7.0). Fraction C: 6 5.97 (dd, J6.5, 15.9 Hz), 5.81 (dd, J8.9, 15.9), 5.38 (m), 3.97 (m), 2.82 (m), 0.974 (t, J 7.5), 0.970 (t, J 7.7); this sample decomposed before running the MS spectrum. Fraction D: m/z

-

Nota 644 (M+),626,620 (M+),602,316,298; 6 5.88 and 5.86 (two poorly resolved dd's), 5.38 (m), 4.90 (m), 4.19 (m), 2.83 (m), 0.97 (t, J 7.4 Hz), 0.95 (t, J 7.4),0.88 (distorted triplet). Fraction E m / z 646 (M+),628, 318, 300; 6 5.96 (dd, J 6.5, 15.8 Hz), 5.78 (dd, J 8.9,15.8), 5.38 (m), 3.97 (m), 2.83 (m), 0.97 (t,J7.5),0.89 (distorted triplet). Fraction F m/z 572 (M+) 554,316,298; b 5.97 (dd, J 6.5, 15.9 Hz), 5.81 (dd, J8.9, 15.9),3.96 (m), 0.97 (t,J7.5),0.88 (t,J 6.6). Fraction G m / z 598 (M+), 580, 572 (M+),554, 318, 316, 300, 298; 6 5.88 (dd, J 5.9, 15.8 Hz), 5.86 (dd, J 8.2, 15.8), 4.90 (m), 4.19 (m), 3.97 (m) (4.W3.97 = 21), 0.97 (t,J 7.5), 0.95 (t,J 7.5). Fraction H: m / z 574 (M+), 556,318,300,b 5.96 (dd, J 6.6, 15.9 Hz), 5.78 (dd, J 8.9, 15.9), 3.97 (m), 0.88 (distorted triplet). Anatomical sections of T. fimbria were obtained as follows. Mantles and cerata of four T. fimbria specimens were separated from the internal organs, and then the latter were subdivided in ovotestis, hepatopancreas, prostate, penis, and seminal receptacle. The anatomical sections were separately extracted and analyzed by TLC. The PLFE mixture was detected in the ovotestis only. The ethereal extract of the ovotestis (28 mg)was purified by TLC as above to give 0.6 mg of a PLFE mixture whose 'H NMR spectrum was similar, where relevant, to that of the mixture extracted from the egg masses. Methanolysis of the PLFE Mixture. Part of the PLFE mixture (3.5 mg)was dissolved in 1mL of anhydrous MeOH, and 10 mg of solid Na&03 was added. After 20 h at room temperature, the MeOH solution was recovered by filtration and aliquots were analyzed by gas chromatography (25-m glam capillary column; SE30 0.5% on C B 110 "C). Identification of the fatty acid methyl esters was made by standard procedures, vs reference compounds (Sigma). The esters identified were the following (percent in the mixture): c149 (7.41, Clel (5.91, Claa (12.1), CIW(8.11, C I , ~(3.41, ClE1 (3.4, Cleo (9.51, C2k4 (arachidonic; LO), (2%. (cis5,8,11,14,17-emosapentaenoic;12.31, Cw. (5.1), Cz2* (cis4,7,10,13,16,19-docosahexaenoic; 14.6). The remaining part of the MeOH solution was submitted to preparative HPLC on reversed-phasecolumn (Spherisorb 5 pm as for the quantitation method, see above), resulting in the isolation, in order of increasing retention time, of PGF,-methyl eater, PGFk-methyl ester, and PGFk-1,151adone. An authentic sample of PGFk-methyl ester was prepared by CH2N2methylation of commercially available PGFk, while PGFk-1,15-lactone was identified by comparison with a synthetic sample.' Prostaglandin Fk methyl ester: MS, m / z (%) 350 (M+ H2O; 2.0), 332 (M+- 2 HZO; 14), 314 (M+ - 3 H2O; 5), 278 ( b m peak); 'H NMR 6 (CDCIS)5.58 (dd, J 6.4, 15.2 Hz; 1H, H-14), 5.51 (dd, J8.5, 15.2; 1H, H-13), 5.41 (m; 2 H, H-5 + H-6),4.20 (m; 1H, H-9), 4.08 (q, J6.4; 1H, H-15),3.98 (m; 1H, H-ll), 3.67 (8; 3 H, OCH3), 1.81 (bd, J 14.3; 1 H, H-loa), 0.89 (bt, J 6.6; 3 H, H-20). Prostaglandin F, methyl ester: MS, m/z (%) 348 (M+ HZO; 1.5), 330 (M+ - 2 HZO; 5), 312 (M+ - 3 HZO; 2), 297 (26), 279 (82), 261 ( b m peak); 'H NMR 6 (CDClJ 5.61 (dd, J 6.0,15.3 Hz,1H, H-14), 5.56 (dd, J9.0,15.3; H-13, overlapped with mother olefhic proton), 5.45-5.32 (three overlapped olefinic protons), 4.21 (m; 1H, H-9),4.16 (q, J6.3; 1H, H-15),4.0 (m; 1H, H-ll),3.67 (8; 3 H, OCHs), 1.81 (bd, J 14.5; 1 H, H-loa), 0.97 (t,J 7.5; 3 H, H-20). Ichthyotoxicity Test. Ichthyotoxicity assays were conducted on the mosquito f i h Gambwia affinis as previously described.4' In each test, six fiihes were placed in distilled water (70 mL) and 0.5 mL of an acetone solution of appropriate concentration of the test compound was added. The toxicity ranking was defined according to ref 6. The results of the testing were the following: 1, toxic at 10 ppm; 2, toxic at 1 ppm; 3, toxic at 5 ppm; 4, not t e s M , 5, 6, and the PLFE mixture, nontoxic at 10 ppm.

Acknowledgment. We are grateful to Dr.G. L. Bundy, Upjohn Co., Kalamazoo, for the generous gift of synthetic PGE2-l,15-lactone and PGF%-l,15-lactone and to Dr.A. Marin Atucha, University of Murcia, and Mias E. Martinez (6) Coll, J. C.; La Barre, S.;Sammarco, P.W.; Williams, W.T.;Baku, G. J. Mar. Ecol. Prog. Ser. 1982,8, 271. (7) Gunthorpe, L.;Cameron, A. M.Mar. Eiol. 1987,94,39.

2911

J. Org. Chem. 1991,56,2911-2912

Cueto-Felgueroso, University of Oviedo, for anatomical dissection of T.fimbria. We also thank Miss C. Minardi, Mr. A. Trabucco, and Mr. G. Villani for technical assistance and Mr. R. Turco for artwork. The NMR spectra and the mass spectra were, respectively, obtained from the ICMIB-NMR Service and from the "Servizio di Spettrometria di Massa del CNR e dell'Universit4 di Napoli"; both staffs are gratefully acknowledged. Registry NO. 4,62410-96-2;5, 132541-84-5;6, 132618-68-9; 7, 55314-49-3; 8, 132564-12-6; 9, 132541-85-6; 10, 132541-86-7; Sacyl-PGF, Cm fatty acid ester, 132564-41-1;9-acyl-PGFs, C140 fatty acid ester, 132541-87-8;11-acyl-PGF, Cm fatty acid ester, 132541-88-9; ll-acyl-PGFz, Cmr fatty acid ester, 132541-89-0; Sacyl-PGFz,Cm fatty acid ester, 132541-90-3;Sacyl-PGF, Cia+ fatty acid ester, 132564-13-7;ll-acyl-PGFS, Ca+ fatty acid ester, 132541-91-4; ll-acyl-PGFz, Clel fatty acid ester, 132541-92-5; 9-acyl-PGFk Cleo fatty acid ester, 132541-93-6;11-acyl-PGF,, Clal fatty acid ester, 132541-94-7;11-acyl-PGF, Cleo fatty acid ester, 132541-958;Sacyl-PGF, Clel fatty acid ester, 132541-96-9; 9-acyl-PGFz,Clel fatty acid ester, 132541-97-0;3, 123314-21-6; 11-acyl-PGF3, Cm4 fatty acid ester, 132564-42-2;11-acyl-PGF, CZEdfatty acid ester, 132541-98-1.

Use of N,"-Dimethoxy-N,N'-dimethylurea as a Carbonyl Dication Equivalent in Organometallic Addition Reactions. Synthesis of Unsymmetrical Ketones Wesley L. Whipple' and Hans J. Reich*

Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706 Received September 12,1990

Many methods for the formation of ketones by the addition of organometallic reagents to C02equivalents appear in the literature? These include the addition of the organometallic reagent to carboxylic acid derivatives,*s direct addition to C02, and reactions employing a transition-metal catalyst.' Even though many of these approaches produced the desired ketones, yields were often low and the reaction conditions were specific and not broadly applicable. A common complicating factor is the concomitant addition of the organometallicreagent to the ketone to produce a tertiary alcohol. Several research groups have introduced ester or amide derivatives in which the formation of tertiary alcohols is not a problem.H These derivatives (e.g., 1) contain a ligand that stabilizes by chelation the tetrahedral intermediate 2 formed by addition of the organometallic reagent. The most effective of these appear to be amides (1) Present address: Nalco Chemical Co., One Nalco Center, Naperville, IL 60563. (2) For reviewe see: Shirley, D. A. Org.React. 1964,8,28. Jorgenson, M. J. Org. React. 1970, 18, 1. (3) Oweley, D. C.; Nelke, J. M.; Bloomfield, J. J. J. Org. Chem. 1973, 38,901. Kende, A. S.;Scholz, D.; Schneider, J. Synth. Commun. 1978, 8,59. Sviridov, A. F.; Ermolenko, M. S.;Yashunsky, D. V.; Kochetkov, M. K. Tetrahedron Lett. 19&3,24,4355. Sato, F.;Inoye, M.; Oguro, K.; &to, M.; Tetrahedron Lett. 1979 20,4303. Kikkawa, I.; Yorifuji, T. Synthesie 1980,877. Wattanasin, S.; Kathawala, F.G. Tetrahedron Lett. 1)84,25,811. (4) Cardellicchio, C.; Fiandaneee, V.; Marcheee, G.;Ronzini, L. Tetrahedron Lett. 1986,26,3595. (5) Mukaiyama, T.; Araki, M.; Takei, H.J. Am. Chem. SOC.1973,95,

-.

AIR.1. --.

(6) Meyere, A. I.; Comins, D. L. Tetrahedron Lett. 1978, 19, 5179. (7) Nahm, S.; Weinreb. S. M. Tetrahedron Lett. 1981,22, 3815. (8) Oater, T. A.; Harris,T. M. Tetrahedron Lett. 1983,24, 1851.

0 RKN'O,

5

I

-

1

2

3

0

I 4

t 5

(1)' or ureas (4,5),9 which use the N-methoxy-N-methyl ligand. Nahm and Weinreb discovered that several Nmethoxy-N-methylamides reacted cleanly with Grignard and organolithium reagents to produce ketones.' For these compounds, the intermediate adduct (2, M = Li, Mg) is stable up to room temperature. The ketone 3 is formed only during the hydrolysis, and the intermediate 2 can actually be submitted to additional chemical manipulation (e.g., deprotonation and alkylation of a remote N,N-dimethylhydrazone present in the moleculelo).

Results and Discussion We report the synthesis of N,N'-dimethoxy-N,"-dimethylurea (4) and its use as a carbonyl dication equivalent. This reagent was also independently developed by Hlasta and The urea 4 was formed in 78% yield from the addition of a solution of bis(trichloromethy1) carbonate (triphosgene) in T H F to N-methoxy-Nmethylamine. (Trichloromethy1)chloroformate (diphosgene) also works well. The addition of 4 to solutions of a variety of aryl, alkyl, alkenyl, and alkynyl organometallic reagents in THF readily produced the amide 1. The reactions were clean and occurred in high yield in all cases except where the formation of the organolithium reagent was difficult (le and If, Table I). The reactions proceeded slowly at -78 "C, but rapidly as the reaction mixtures were warmed to 22 "C. A reaction mixture containing 2-lithio-&methylthiophene and 4 at -78 OC was quenched with a 1 M aqueous/MeOH solution of NH4Cl after 10 min and gave only 2% of la, whereas an analogous reaction mixture that was warmed to room temperature and then quenched gave a near quantitative conversion. The formation of the ketone 3 from the addition of RLi or RMgBr reagents to the amide 1 is well precedented' but for completeness is illustrated for compounds la, Id, and le. The urea 4 provides an excellent route to ketones and should prove useful in many synthetic applications. It is easily prepared and purified, is stable for long periods of time, and reacts cleanly with RLi and RMgBr reagents. It should be particularly useful when the intermediate amide 1 cannot be synthesized by traditional methods because of the instability of the parent carboxylic acid. Experimental Section General Procedures. Unless otherwise state, all 'H NMR spectra were measured with reference to CHC&(6 7.24) or TMS (6 0.0). The CDCls triplet (6 77.0) was used as reference for lac NMR. Infrared (IR) spectra were taken of neat liquids, unless otherwise stated, between NaCl plates. All elemental analyses were performed by Galbraith Laboratories. Kugelrohr distillation refers to bulb-to-bulb distillation (bath temperatures are reported). Diethyl ether and tetrahydrofuran (THF) were freshly distilled from sodium benzophenone ketyl. (9) Hlasta, D. J.; Court, J. J. Tetrahedron Lett. 1989, 30,1773. (10) Evans, D. A.; Bender, S. L.; Morris, J. J. Am. Chem. SOC.1988, 110,2506.

0022-3263/91/1956-2911$02.50/00 1991 American Chemical Society